1. Targeting Insulin Receptor with a Novel Internalizing Aptamer
Margherita Iaboni, Raffaela Fontanella, Anna Rienzo, Maria Capuozzo, Silvia Nuzzo, Gianluca Santamaria, Silvia Catuogno, Gerolama Condorelli, Vittorio de Franciscis, Carla Lucia Esposito 
Molecular Therapy - Nucleic Acids 
Volume 5, (January 2016)
DOI: /mtna
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Cell-internalizing SELEX. (a) Scheme of the cell-internalizing protocol. We performed two cycles in which the G13 pool was incubated with U87MG target cells. Unbound aptamers were discarded and cells were treated with proteinase K for 30 minutes to remove cell surface-bound aptamers. Internalized aptamers were recovered by RNA extraction and RT-PCR. (b) Dendrogram by MAFFT analysis of the 50 individual sequences cloned after the two rounds of internalization. The most represented sequences are boxed. (c) Scattering plot of the 100 most abundant aptamers obtained by Illumina deep sequence analyses of the two cycles of internalization. The top five aptamers are indicated. SELEX, Systematic Evolution of Ligands by Exponential enrichment; RT-PCR, reverse transcription polymerase chain reaction.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
GL56 aptamer targets IR. (a) Lysates from U87MG left untreated (Ctrl) or treated with GL56 and, then, stimulated with 20% FBS were subjected to Human Phospho-RTK arrays. Left, RTK arrays. Boxes indicate phospho-IR (black boxes) and phospho-IGF-1R (white boxes) signals. Right, Quantization performed with ImageJ (v1.46r) of phospho-IR and phospho-IGF-1R signals. (b) U87MG cells were transfected with a siRNA specific for IR (IR siRNA) or a control siRNA (Ctrl siRNA). Left, GL56 or a control unrelated aptamer (Ctrl-Apt) binding ability was measured by RT-qPCR at 200 nmol/l concentration. Right, IR and IGF-1R expression levels were analyzed by immunoblotting with IR beta (IRβ) and IGF-1R beta (IGF-1Rβ) specific antibodies. Anti-αTubulin (αTub) antibody was used to confirm equal loading. (c) GL56 or G0 starting pool (used as control) binding to the IR purified extracellular domain (EC-IR) was measured by filter binding coupled to RT-qPCR. (d) The interaction of biotinylated GL56 with EC-IR was analyzed by ELASA assay. An antibody specific for IR-α (anti-IRα) was used as positive control. In a-d error bars depict standard deviation values on analytical replicates. IGF-1R, insulin-like growth factor receptor 1; IR, insulin receptor; FBS, fetal bovine serum; RT-qPCR, reverse transcription quantitative polymerase chain reaction; ELASA, enzyme-linked apta-sorbent assay.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
GL56 binging specificity. (a) and (b) Aptamer-mediated pull-down. Indicated cell lines were incubated with the biotinylated unrelated aptamer or GL56, cell lysates were purified on streptavidin beads and immunoblotted with anti-IRβ or anti- IGF-1Rβ antibodies. 20 µg of total cell extracts (Tot Extr) were loaded as references. (c) Binding by RT-qPCR of control unrelated aptamer (Ctrl-Apt) or GL56 aptamer on U87MG and T98G cells. Error bars depict standard deviation values on analytical replicates. IGF-1R, insulin-like growth factor receptor 1; RT-qPCR, reverse transcription quantitative polymerase chain reaction.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
GL56 internalization. (a, b) Confocal microscopy analysis. (a) Alexa-488 labeled-GL56 was incubated on U87MG or T98G for 30 minutes. (b) Alexa-488 labeled-GL56 or G0 pool (as control) were incubated on U87MG for 15 minutes (upper panel) and single confocal Z layers (of 1 µm each) were acquired (lower panel). In (a,b) Nuclei stained with DAPI are shown in blue. Scale bars: 10 µm. (c) Total bound or internalization of GL56 onto U87MG was measured by cell binding with radiolabeled aptamer. The background values obtained with the G0 pool were subtracted from the values obtained with the GL56. The percentage of internalized aptamer relative to total bound is indicated. Error bars depict standard deviation values on analytical replicates. (d) GL56 serum stability were measured by incubating the aptamer at 4 μmol/l in 90% human serum for indicated times. At each time point, RNA-serum samples were collected and evaluated by electrophoresis with 15% denaturing polyacrylamide gel. Gel was stained with ethidium bromide. DAPI, 4',6-Diamidino-2-Phenylindole.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
GL56 inhibitory activity. (a) U87MG cells were serum starved overnight, left untreated or pretreated for 3 hours with 400 nmol/l GL56 aptamer or control aptamer (Ctrl-Apt) and, then, stimulated for 5 minutes with 100 nmol/l insulin alone or in presence of 400 nmol/l each aptamer. Cell lysates were immunoprecipitated with anti-(phospho)-tyrosine (pTyr) antibody and immunoblotted with anti-IRβ or were immunoblotted with anti-IRβ antibody. (b) Serum-starved U87MG were left untreated or pretreated for 3 hours with 400 nmol/l GL56 aptamer and stimulated for 5 minutes with 100 nmol/l insulin alone or in presence of 400 nmol/l GL56. Phopsho-AKT (pAKT), phospho-ERK1/2 (pERK), and phopsho-IRS1 levels were monitored by immunoblotting. Filters were probed with AKT, extracellular signal-regulated kinase (ERK) or IRS1 antibodies to confirm equal loading. (c) Serum starved U87MG were grown in Dulbecco's modified essential medium (DMEM) containing 0.5% fetal bovine serum (FBS) and 100 nmol/l insulin either in absence or in presence of 400 nmol/l GL56 or G0 pool, used as negative control. Treatments were renewed each 24 hours. Cell viability was measured following 48 hours.
Molecular Therapy - Nucleic Acids 2016 5, DOI: ( /mtna )
Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions